The authors thank Dara Frank and Carrie Harwood for their generous contribution of strains. University of Wisconsin Milwaukee Research Foundation holds patent no. 8,354,451; with claims broadly directed to treating or inhibiting the progression of infection of a microorganism in a patient by administering a 4-hydroxyphenylpyruvate dioxygenase-inhibiting compound such as 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). Inventors are Graham Moran and Pang He. This research was supported by the National Institutes of Health (R00-GM083147). The University of Washington P. aeruginosa transposon mutant library is supported by NIH P30 DK089507.

1. Preparação de Meios de Cultura, antibióticos e 2- [2-nitro-4- (trifluorometil) benzoil] -1,3-ciclo-hexanodiona (NTBC) <ol><li> Adicione caldo LB (1% de triptona, 0,5% de extracto de levedura, 0,25% de NaCl em H 2 O) e alíquota em volumes apropriados. Esterilizar em autoclave. Guarde-o em temperatura ambiente. </li><li> Adicione 100 ml de agar LB (1% de triptona, 0,5% de extracto de levedura, 0,25% de NaCl, 1,5% de agar em H2O) em 250 ml de frascos. Esterilizar em autoclave e armazenar à temperatura ambiente. Certifique-se de que o agar é derretido antes de verter em placas. NOTA: Os frascos contendo 100 ml de LB agar irá originar 4 placas. A quantidade de agar LB pode ser alterada para corresponder ao número de placas necessárias para o ensaio. </li><li> Adicione PBS (NaCl 137 mM, KCl 2,7 mM, 10 mM de Na 2 HPO 4, KH 2 mM 2 PO 4) 16. Esterilizar em autoclave ou filtração e armazenamento à temperatura ambiente. </li><li> Preparar as soluções de antibióticos para a gentamicina, a canamicina, umad tobramicina. <ol><li> Prepare concentrações banco de antibióticos apropriados para P. aeruginosa estirpes contendo 100 mg / ml de gentamicina, 30 mg / ml de canamicina, e 10 mg / ml de tobramicina. Dissolver os antibióticos em água, Esterilizar por filtração (0,2 mm), e armazenar a 4 ° C. Alterar os antibióticos e concentrações, dependendo da bactéria em estudo. </li></ol></li><li> Prepare as soluções estoque NTBC. Dissolve-se 10 mg de NTBC em 400 ul de DMSO. Isto produz uma concentração de 75,9 mM NTBC. Loja soluções estoque NTBC a -20 ° C. Soluções descongelar à temperatura ambiente, conforme necessário. NOTA: Diferentes fontes de NTBC têm diferenças de solubilidade. Determinar o veículo apropriado em que para dissolver o NTBC com base nas recomendações do fabricante e ajustar Passo 1.5 em conformidade. </li></ol> 2. NTBC Titulações de Estirpes Bacterianas <ol><li> Defina-se culturas nocturnas das estirpes a serem testadas. Adicionar 2 ml de caldo LB a 16 x 150 mm tubo de ensaios (um por cepa) e inocular com uma colônia isolada de cada estirpe. Incubar durante a noite a 37 ° C com arejamento num agitador rotativo de cultura de tecidos em uma incubadora de ar. </li><li> No dia seguinte, preparar titulações de NTBC em caldo LB. Use uma primeira série 0-900 mM NTBC uma vez que diferentes cepas têm diferenças na sensibilidade à NTBC. <ol><li> Adicionar 1 ml de caldo LB a 4 para 5 tubos de ensaio (16 x 150 mm) por estirpe. </li><li> Adicionar a solução estoque NTBC (75,9 mM), para os tubos de ensaio (16 x 150 mm) numa gama de concentrações. Ver Tabela 1 para as concentrações e volumes NTBC imagens correspondentes a adicionar a 1 mL de caldo de LB. </li></ol></li><li> Medir a OD 600 das culturas durante a noite. Lave as culturas antes de tomar OD 600 leituras para eliminar pyomelanin presente na mídia. <ol><li> Lavam-se as culturas por centrifugação de 1 ml de cultura em uma microcentrífuga a 16000 xg durante 2 min. Remover o sobrenadante e as células peletizadas com frouxamenteuma micropipeta e ressuspender o pellet celular sólido em 1 ml de LB. </li></ol></li><li> Inocular tubos de titulação em OD 600 de 0,05. Calcula-se a quantidade necessária da cultura lavou-se para inocular os tubos. NOTA: Use as culturas lavadas para inoculações desde pyomelanin não deve estar presente. </li><li> Incubar os tubos de titulação durante aproximadamente 24 horas a 37 ° C com arejamento usando um tecido rotador cultura numa incubadora de ar. </li><li> Fotografar os tubos de titulação e comparar a produção de pigmento dentro e entre as cepas para determinar a quantidade de NTBC de usar para MIC e estresse oxidativo ensaios. Use OD 600 leituras para determinar a quantidade de pyomelanin na célula livre sobrenadante da cultura e para determinar a densidade celular. Observação: A razão OD 600 de pyomelanin no sobrenadante da cultura de células pode ser calculada para quantificar as diferenças na produção pyomelanin após o tratamento com NTBC. </li></ol> 3. Antibiótico Inhibi mínimoConcentração (MIC) Ensaio tory em placas de 96 poços <ol><li> Configure culturas durante a noite das estirpes a ser testado em LB com e sem NTBC. NOTA: Este protocolo é descrito usando o nível representante de 300? M NTBC. O nível apropriado de NTBC a ser utilizado é determinado no passo 2.6. <ol><li> Adicionar 300 ^ M a 2 ml NTBC LB. Adicionar um volume equivalente de veículo (DMSO) a 2 ml de LB para a condição não NTBC. </li><li> Usando palitos estéreis, inocule os tubos com uma colônia de bactérias isoladas. Haverá uma cultura com NTBC e uma cultura sem NTBC para cada estirpe. Incubar durante a noite a 37 ° C com arejamento usando um tecido rotador cultura numa incubadora de ar. </li></ol></li><li> No dia seguinte, fazer LB + NTBC e soluções mestre LB + DMSO para a criação do ensaio MIC. Adicionar NTBC a uma concentração de 600 uM como este será diluído duas vezes quando inoculo é adicionado, dando origem a uma concentração final de 300 uM. <ol><li> Para testar um antibiótica para uma estirpe, adicionar 600 | iM NTBC a 2 ml de LB e misturar para tornar a solução NTBC mestre. Adicionar um volume equivalente de veículo (DMSO) a 2 ml de LB e misturar para fazer a solução não NTBC mestre. Usar estas soluções para criar soluções-mãe de antibióticos, bem como para a criação de séries de diluição em placas de 96 poços. NOTA: As formulações de solução mestre trará solução extra para contabilizar erros de pipetagem. As soluções mestre pode ser escalada para cima ou para baixo, conforme necessário, dependendo do número de antibióticos e estirpes testadas. </li></ol></li><li> Preparar as soluções de antibióticos nas soluções mestre LB + NTBC ou LB + DMSO. NOTA: A concentração do antibiótico na Estas soluções devem ser o dobro da concentração final desejada. Deve ser feita solução suficiente para transferir 100 ul de quatro poços de uma placa de 96 poços. <ol><li> Prepare gentamicina +/- solução estoque NTBC a 64 ug / ml. Para tornar esta solução, adicionar 0,288 ul de estoque de gentamicina (100 mg / mL) a 450jil de LB + NTBC ou mestre solução de LB + DMSO. NOTA: A concentração máxima de gentamicina para P. aeruginosa PAO1 é de 32 ug / ml. </li><li> Faça a canamicina +/- solução estoque NTBC a 256 ug / ml. Para tornar esta solução, adicionar 3,84 l de estoque canamicina (30 mg / ml) a 450 ul soluções mestre LB + NTBC ou LB + DMSO. NOTA: A concentração máxima de canamicina para P. aeruginosa PAO1 é de 128 ug / ml. </li><li> Prepare tobramicina +/- solução estoque NTBC a 8 ug / ml. Para tornar esta solução, adicionar 0,36 l de estoque tobramicina (10 mg / ml) a 450 ul soluções mestre LB + NTBC ou LB + DMSO. NOTA: Para P. aeruginosa PAO1, a concentração máxima de tobramicina é de 4 ug / ml. NOTA: As concentrações de antibióticos e pode ser regulado para as bactérias a serem testadas. </li></ol></li><li> Adiciona-se 100 ul de cada solução de antibiótico 2x para poços quatro numa placa de 96 poços. Coloque essas soluções em linha A. Para example, a gentamicina, deve ser colocado em A1 a A4, canamicina devem ser colocados em A5 a A8 e tobramicina deve ser colocado em meio A9 A12. Ver Figura 1A um diagrama de uma placa de 96 poços configurado. NOTA: antibióticos múltiplos pode ser testado em um prato, mas apenas uma estirpe deve ser testado por placa para eliminar o potencial de contaminação cruzada de outras estirpes. </li><li> Adicionar 50 ul da solução de mestre de LB + NTBC ou LB + DMSO a linha B a H da placa de 96 poços. Certifique-se que uma placa é LB + NTBC e um prato é LB + DMSO. Veja a Figura 1A. <ol><li> Use de LB + NTBC para os antibióticos em LB + NTBC. Use de LB + DMSO para os antibióticos em LB + DMSO. </li></ol></li><li> Usando uma micropipeta realizar diluições em série duas vezes dos antibióticos através da transferência de 50 mL da solução de linha A para a linha B. Misturar a solução, mudar as pontas de pipetas, e transferir 50 ul da solução a partir da linha B para a linha C para Repetir o remainilinhas GN. Depois de se diluir linha L, remover 50 ul da solução a partir dessa linha e descarte. Utilize linha H como um antibiótico sem controle para o crescimento bacteriano. Ver Figura 1B. NOTA: Cada poço nas filas A a G contém agora 50 uL de antibiótico em LB + NTBC ou LB + DMSO a 2X a concentração final desejada. Fila H contém LB + NTBC ou LB + DMSO sem antibióticos. </li><li> Medir a OD 600 das culturas durante a noite. Lave todas as culturas antes de tomar OD 600 leituras para eliminar pyomelanin presente na mídia. <ol><li> Lavam-se as culturas por centrifugação de 1 ml de cultura em uma microcentrífuga a 16000 xg durante 2 min. Remover o sobrenadante com uma micropipeta e ressuspender o pellet celular em 1 ml de LB. </li></ol></li><li> Dilui-se as culturas durante a noite para 2.75x10 5 ufc / mL em LB. NOTA: Assume-se que uma unidade DO600 é o equivalente de 1x10 9 CFU / ml para P. aeruginosa. OD para as conversões de UFC / mL podem ser different em outras bactérias. </li><li> Adicionar 50 ul da cultura bacteriana diluído ao poço adequado. NOTA: As culturas são crescidas em NTBC deve ser adicionada aos poços contendo NTBC e culturas cultivadas em DMSO deve ser adicionado às cavidades contendo DMSO. Ver Figura 1B. <ol><li> Adicionar bactérias a três poços para cada estirpe e concentração do antibiótico. Adicionar 50 ul de LB para o quarto poço para actuar como um controlo para a contaminação bacteriana. Ver Figura 1B. </li><li> Usar uma pipeta de multi-canal para inocular os poços. Assegurar que pipeta pontas estão perto do fundo dos poços, quando a adição de inoculo para evitar a contaminação de poços vizinhos. NOTA: A adição de cultura bacteriana aos poços irá diluir as concentrações de antibiótico e NTBC duas vezes. </li></ol></li><li> Cobrir as placas de 96 poços com parafilme e incubar a cerca de 24 horas a 37 ° C. Incubar as placas de 96 poços estaticamente, numa incubadora de ar. </li><li> Examinaras placas para o crescimento bacteriano nas cavidades. A MIC é a concentração mais baixa de antibiótico em que nenhum crescimento bacteriano é visto para todas as três repetições de cada estirpe. <ol><li> Examine visualmente a placa de crescimento ou ler usando um conjunto leitor de placas de DO600. </li></ol></li></ol> 4. Ponto Ensaio Placa de resposta ao estresse oxidativo <ol><li> Defina-se culturas nocturnas das estirpes a ser testado em meio LB com ou sem NTBC tal como descrito na etapa 3.1. </li><li> No dia seguinte, para preparar as placas de agar LB contendo H 2 O 2 como um estressor oxidativo. Uma gama de H 2 O 2 concentrações de 0 a 1 mM é um bom ponto de partida. <ol><li> Derreter os frascos de LB-agar. Super meios para aproximadamente 50 ° C à temperatura ambiente. </li><li> Adicionar H 2 O 2 directamente sobre o suporte arrefecidos nas concentrações desejadas. Frascos agite para misturar. Ver Tabela 2 para as concentrações de H 2 O 2e volumes de concentrado H 2 O 2 a acrescentar. Estes valores baseiam-se em 100 ml de LB com agar. </li><li> Verter em placas imediatamente após a adição de H 2 O 2 e a superfície da chama para remover as bolhas. O rendimento é de 4 placas por 100 ml de LB com agar. Marcar as placas com a concentração de H 2 O 2. </li><li> Colocar as placas descoberto numa câmara de fluxo biológico com o ventilador corrida durante 30 min para remover o excesso de humidade das placas. NOTA: Use as placas no mesmo dia em que são preparados. Não fazer isso pode resultar em dados inconsistentes. NOTA: factores de stress oxidativo, tais como o paraquat pode ser substituído por H 2 O 2 neste ensaio. As concentrações utilizadas para outros factores de stress oxidativo pode ser diferente do que os usados ​​para a H 2 O 2. </li></ol></li><li> Lavar e medir a DO 600 das culturas durante a noite, tal como descrito na etapa 3.7. </li><li> Normalizar o OD 600 de todo o cu durante a noiteltures para o menor valor para o conjunto de estirpes que está sendo testado. P. aeruginosa tem geralmente uma DO600 de aproximadamente 2,5, quando crescidas durante a noite em LB ou LB + NTBC + DMSO. <ol><li> Determinar o volume de cultura necessárias para diluir a cultura para o menor OD 600 num volume total de 1 ml. Por exemplo, se uma cultura tem um OD 600 de 3 e menor do OD 600 para o conjunto de estirpes é de 2,5, executar o seguinte cálculo: (2,5) (1 ml) = (3) (X). x = 0.833 ml. 0,833 ml de cultura vai ser colocado num tubo de microcentrífuga. </li><li> Calcular a quantidade de LB + NTBC (300 uM) ou DMSO de LB + necessária para trazer o volume de cultura para 1 ml. Para o exemplo no passo 4.4.1, a quantidade de LB + NTBC ou LB + DMSO adicionado à cultura seria 0.167 ml (1 ml de volume total de 0,833 ml - de cultura). Adicione as soluções concentradas de LB + NTBC e LB + DMSO para utilizar para estes diluições com base no volume necessário para diluir todas as estirpes. </li><li> Misture a cultura e LB + NTBC ou LB + DMSO em vórtice. </li></ol></li><li> Para manter as culturas numa concentração constante de NTBC ou DMSO, realizar diluições em série de dez vezes das culturas durante a noite em PBS + normalizados NTBC ou PBS + DMSO. <ol><li> Faça as soluções concentradas de PBS + NTBC e PBS + DMSO. Para um conjunto de diluições de uma estirpe, misturar 300 NTBC uM ou um volume equivalente de DMSO com PBS para se obter um volume total de 720 ul. Escalar esses stocks cima ou para baixo, dependendo de quantas cepas são testadas. </li><li> Etiqueta para tubos de microcentrífuga através de diluições 10 -1 10 -7 série. Adicionar 90 ul de PBS + NTBC ou PBS + DMSO para os tubos apropriados. Use PBS + NTBC para as estirpes cultivadas em LB + NTBC e usar PBS + DMSO para as estirpes cultivadas em LB + DMSO. </li><li> Adicionar 10 ul de cultura para o tubo 10 -1 de diluição apropriado. Misturar em vortex e transferir 10 ul da diluição 10 -1 para o tubo de diluição 10 -2. Repetir até que todas as diluições foram Performou. Alterar pontas de pipeta entre diluições. </li></ol></li><li> Spot 5 ul de 10 -3 a 10 -7 através diluições em placas de LB + H 2 O 2 placas em duplicado para cada estirpe. Use uma ponta de pipeta se manchas são banhados de mais diluída para menos diluído (10 -7 a 10 -3). Não ponta ou incline a placa até que o líquido tenha secado na placa. </li><li> Incubar as placas durante 24-48 horas a 37 ° C (incubadora de ar), dependendo da estirpe. NOTA: P. aeruginosa PAO1 terá bons colônias porte sobre LB após 24 horas de incubação. Incubar estirpes até que colónias têm aproximadamente o mesmo tamanho que PAO1. </li><li> Fotografar as placas usando uma câmera CCD acima de um transluminador. Opcionalmente, editar as fotos para o contraste e cultura para o mesmo tamanho. Contar o número de colónias em cada local para determinar alterações na sensibilidade ao estresse oxidativo. </li></ol>

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<li>Khajo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protection+of+Melanized+Cryptococcus+neoformans+Lethal+Dose+Gamma+Irradiation+Involves+Changes+in+Melanin%27s+Chemical+Structure+and+Paramagnetism%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Protection of Melanized Cryptococcus neoformans Lethal Dose Gamma Irradiation Involves Changes in Melanin's Chemical Structure and Paramagnetism.</a> PLoS ONE. 6, e25092(2011).</li>
<li>Hancock, R. E. W. Hancock Laboratory Methods. , University of British Columbia, Department of Microbiology and Immunology. British Columbia, Canada. Available from:
	 
 <a target="_blank" href="http://www.cmdr.ubc.ca/bobh/methods.htm">http://www.cmdr.ubc.ca/bobh/methods.htm</a> (2015).</li>
</ol>

The authors declare that they have no competing financial interests.

The NTBC titration method described in this protocol will allow the user to determine if NTBC can reduce or eliminate pyomelanin production in bacteria, and determine the concentration of NTBC required. The most critical step in the NTBC titration assay is determining the range of NTBC concentrations to use in the assay. Different strains of P. aeruginosa have different sensitivities to NTBC, and laboratory strains may be more sensitive to NTBC than clinical isolates12 (Figure 2). The...

Pseudomonas aeruginosa is a Gram negative bacterium that produces a variety of pigments including pyomelanin, a red-brown pigment that helps provide protection from oxidative stress1-4 and binds a variety of compounds, including aminoglycoside antibiotics5-7. Pyomelanin production is caused by a defect in the tyrosine catabolism pathway4,8, either through deletions or mutations of the gene encoding homogentisate 1,2-dioxygenase (HmgA)1,9 or through imbalances in the various enzymes in the pathway10. Homogentisate accumulates due to inactivation of HmgA, and is secreted and oxidized to form pyomelanin11. Production of pyomelanin can be abolished or reduced in a dose dependent manner through treatment with the herbicide 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)12, which inhibits 4-hydroxyphenylpyruvate dioxygenase (Hpd) in the tyrosine catabolism pathway13. Hpd is required for the formation of homogentisate, and therefore pyomelanin11. 
We describe in detail three techniques that were important in our studies of NTBC treatment of pyomelanin producing strains of P. aeruginosa. These techniques include titration of NTBC to determine the concentrations that will abolish or reduce pyomelanin production in laboratory and clinical pyomelanin producing strains, determination of the minimum inhibitory concentration (MIC) of antibiotics when bacteria are treated with NTBC, and the resulting sensitivity to oxidative stress with NTBC treatment.
The titration assay we developed serves two purposes. First, the assay will allow the user to determine if NTBC can abolish or reduce pyomelanin production in the bacterium being studied and at which concentrations. This will allow the user to determine sensitivity to NTBC, since different strains of bacteria may have different sensitivities to this compound, as observed in P. aeruginosa12. Second, the NTBC titration assay will allow the user to determine the appropriate concentration of NTBC to use in subsequent assays, such as antibiotic MIC and oxidative stress response assays, if the goal is to abolish or reduce pyomelanin production and determine the effects of pigment reduction. 
The titration assay works because a visible difference in pyomelanin production can be seen in strains treated with NTBC and the differences in pyomelanin production are dose dependent12. Additionally, this technique can be applied to the study of other compounds that may eliminate or enhance pigment production in bacteria.
Antibiotic MICs are used to determine the sensitivity of bacteria to antibiotics. There are several methods to determine MICs, including agar dilution plates and broth dilutions14. Broth dilutions can be performed in small test tubes or in a 96-well microtiter plate. The microtiter plate method of MIC determination described herein will allow the user to test a wide range of antibiotics using a minimum of resources. The assay provides reproducibility as well as flexibility in the number of antibiotics and strains tested by this method. Additionally, with the incorporation of NTBC in the assay, the user can determine if elimination or reduction of pyomelanin production alters antibiotic sensitivity in bacteria that produce pyomelanin.
Bacterial response to oxidative stress can be tested in several ways. The most common methods described are either viable counts of bacteria subjected to oxidative stress for a period of time1, or oxidative stress disc diffusion assays15. These methods tend to use high concentrations of oxidative stressors to examine the effects of oxidative stress in bacteria and results can be quite variable between biological replicates. The viable count assay also tends to use more agar plates than the other methods. The spot plate assay we describe uses low concentrations of H2O2 and allows the user to test the oxidative stress response of multiple strains using a minimum of plates. The assay is also consistently reproducible between technical and biological replicates. As pyomelanin is involved in resistance to oxidative stress, the incorporation of NTBC in the assay allows the user to determine the effects of elimination of pyomelanin production on oxidative stress resistance.

<table><tbody><tr><td>2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)</td><td>Sigma-Aldrich</td><td>SML0269-50mg</td><td>Also called nitisinone.&amp;nbsp; Soluble in DMSO.</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>216763-100ML</td><td>30 wt. % in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Stabilized.</td></tr><tr><td>Gentamicin</td><td>Gold Bio</td><td>G-400-100</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Tobramycin</td><td>Sigma-Aldrich</td><td>T4014-100MG</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr></tbody></table>

methods to inhibit bacterial pyomelanin production and determine the corresponding increase in sensitivity to oxidative stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Titulações NTBC As titulações NTBC foram utilizados para determinar se NTBC foi capaz de reduzir a produção em P. pyomelanin aeruginosa, e também identificar a concentração de NTBC que elimina ou reduz a produção pyomelanin para utilização em ensaios adicionais. Pode haver variações nos níveis de pyomelanin produzida em diferentes repetições, mas as tendências gerais permanecer constante. O ensaio de titulação NTBC também poderia ser modificado para te...

Watch this Scientific Journal Video about Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress at JoVE.com

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Métodos para inibir bacteriana Pyomelanin Produção e determinar o correspondente aumento de sensibilidade ao estresse oxidativo

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

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Research

JoVE Journal

Immunology and Infection

8.2K visualizações.  University of Wisconsin Milwaukee. Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Vídeo: Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

Abordagens para avaliação de estresse oxidativo toxicológico usando sensores Fluorogenic geneticamente codificado de imagem

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Pesquisa do câncer

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

Trans-Plasma da membrana, transporte de elétrons por Myotubes de C2C12 de medição

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

Bioquímica

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

Quantificação de hipopigmentação atividade In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

A indução farmacológica de melanina epidérmica e proteção contra queimaduras solares em um modelo humanizado Rato

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Medicina

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

Detecção de Espécies totais de oxigênio reativo em células aderentes por 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

<meta charset="utf8"></head><body>Bioprocesso adsortivo melhora o rendimento da melanina de Pseudomonas stutzeri

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

Bioprocesso adsortivo melhora o rendimento da melanina de Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

Engenharia

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Detectando, visualizar e quantificar a geração de espécies reativas de oxigênio em um modelo de sistema Amoeba

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Biologia

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Indução e Análise do Estresse Oxidativo em Células Epiteliais de Pigmento Epitelial Humano Transposon-Transfected Da Bela Adormecida

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Avaliação da produção de sideróforos a partir de isolados de Pseudomonas aeruginosa

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification.
The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Autor em destaque: Quantificando sideróforos e pioquelina para controle de infecções 

This protocol provides both qualitative and quantitative analyses of total siderophores, pyoverdine, and pyochelin from Pseudomonas aeruginosa.

Análise Qualitativa e Quantitativa da Produção de Siróforos de Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such ...

Métodos para inibir bacteriana Pyomelanin Produção e determinar o correspondente aumento de sensibilidade ao estresse oxidativo

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